The domestication of rice has been a very important factor in development of civilization in many parts of the world. Rice is intimately involved in the culture, as well as the food and economy, of many societies. For example, according to folklore, when the Kachins of northern Myanmar (Burma) were sent forth from the center of the Earth, they were given the seeds of rice. Rice is an integral part of their creation myth and remains today as their leading crop and most preferred food. In Bali, it is believed that the Lord Vishnu caused the Earth to give birth to rice, and the God Indra taught the people how to raise it. In both tales, rice is considered a gift of the gods, and even today in both places, rice is treated with reverence.
Chinese myth, by contrast, tells of rice seeds being brought to hungry flood survivors on the tail of a dog. The people planted these seeds, rice grew, and hunger disappeared. Throughout China today, tradition holds that xe2x80x9cthe precious things are not pearls and jade but the five grainsxe2x80x9d, of which rice is first.
According to Shinto belief, the Emperor of Japan is the living embodiment of Ninigo-no-mikoto, the god of the ripened rice plant. While most modern Japanese may intellectually dismiss this supernatural role, they cannot deny the enormous cultural importance of rice on life in their country - and so it is in much of the rice world (Huke, R. E. and E. H. Huke [1990] xe2x80x9cRice: Then and Nowxe2x80x9d, International Rice Research Institute).
A greater understanding of rice and an enhanced ability to develop improved phenotypes would be of great value to mankind. Also, of great value to mankind would be improved methods of controlling and directing gene expression generally in eukaryotes, and particularly in plants.
Cultivated rices belong to two species, O. sativa and O. glaberrima. Of the two, O. sativa is by far the more widely utilized. O. sativa is a complex group composed of two forms endemic to Africa but not cultivated, and a third from, O. rufipogon, having distinctive partitions into South Asian, Chinese, New Guinean, Australian, and American forms.
Gene expression in rice, as well as other cells, is a biological function that may be regulated by the cellular processes involved in transcription. During transcription, a single-stranded RNA complementary to the DNA sequence to be transcribed is formed by the action of RNA polymerases. Initiation of transcription in eukaryotic cells is regulated by complex interactions between cis-acting DNA motifs, located within the gene to be transcribed, and trans-acting protein factors. Among the cis-acting regulatory regions are sequences of polynucleotides, termed promoters, enhancers or repressors that are located upstream, or downstream in the case of some elements, to the transcription initiation site. Promoters usually consist of proximal elements (e.g., TATA box) and more distant elements (e.g. CCAAT box). Enhancers are cis-acting DNA motifs that are located further up- and/or down-stream from the initiation site.
Both promoters and enhancers are generally composed of several discrete, often redundant elements, each of which may be recognized by one or more trans-acting regulatory proteins, known as transcription factors. Regulation of the complex patterns of gene expression observed both spatially and temporally, in all developing organisms, is thought to arise from the interaction of enhancer- and promoter-bound, general and tissue-specific transcription factors with DNA (Izawa et al., 1993; Menkens et al., 1995).
The ability to specifically inhibit gene function in a variety of organisms utilizing antisense RNA or ds RNA-mediated interference is well known in the fields of molecular biology (see for example C. P. Hunter, Current Biology [1999] 9:R440-442; Hamilton et al., [1999] Science, 286:950-952; and S. W. Ding, Current Opinions in Biotechnology [2000] 11:152-156, hereby incorporated by reference in their entireties). dsRNA (RNAi) typically comprises a polynucleotide sequence identical or homologous to a target gene (or fragment thereof) linked directly, or indirectly, to a polynucleotide sequence complementary to the sequence of the target gene (or fragment thereof). The dsRNA may comprise a polynucleotide linker sequence of sufficient length to allow for the two polynucleotide sequences to fold over and hybridize to each other; however, a linker sequence is not necessary. The linker sequence is designed to separate the antisense and sense strands of RNAi significantly enough to limit the effects of steric hindrances and allow for the formation of dsRNA molecules and should not hybridize with sequences within the hybridizing portions of the dsRNA molecule.
The specificity of this gene silencing mechanism appears to be extremely high, blocking expression only of targeted genes, while leaving other genes unaffected. A recent example of the use of RNAi to inhibit genetic function in plants used Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana (Chuang, C.-F. and E. M. Meyerowitz [2000], Proc. Natl. Acad. Sci. USA 97:4985-4990). Chuang et aL describe the construction of vectors delivering variable levels of RNAi targeted to each of four genes involved in floral development. Severity of abnormal flower development varied between transgenic lines. For one of the genes, AGAMOUS (AG), a strong correlation existed between declining accumulation of mRNA and increasingly severe phenotypes, suggesting that AG-specific endogenous mRNA is the target of RNAi.
For the development of transgenic plants with desirable traits, constitutive promoters, tissue and organ specific promoters, and cell type specific promoters are required to drive most of the transgenes. The most widely used constitutive plant promoter is derived from the cauliflower mosaic virus. Therefore, there is an urgent need to discover other tissue specific, organ specific, cell specific and constitutive promoters for transgenic applications.
The present invention provides polynucleotides which encode useful proteins and/or are involved in regulation of gene expression. In a preferred embodiment, the isolated polynucleotides of the subject invention are useful in the modification of gene expression in plants. In a specific embodiment, these sequences can be used to modify gene expression in rice. Specifically exemplified herein are sequences which are particularly applicable to gene expression in rice roots.
In one embodiment, the subject invention provides 5xe2x80x2 cis regulatory DNA sequences isolated from rice (Oriza sativa). These sequences can be used in the modification of gene activation and/or expression in eukaryotes, particularly in rice and in other monocots. Promoters provided herein can be used in the modification of expression of genes by virtue of their role as components of the cellular activation and transcription apparatus. Many of these promoters are xe2x80x9ctissue specificxe2x80x9d. As would be understood by one skilled in the art, these promoters can be used to preferentially express gene product in a particular tissue.
The isolated polynucleotides of the subject invention are useful in the modification of gene expression in plants, since both tissue- and temporal- specific gene expression patterns have been shown to be initiated and controlled by promoters during the natural development of a plant. Thus, targeting of these genes can be exploited in the process of developing desirable plant phenotypes.
Purifed nucleotide sequences of this invention have numerous applications in techniques known to those skilled in the art of molecular biology having the benefit of the instant disclosure. These techniques include their use as hybridization probes, for chromosome and gene mapping, in PCR technologies, and in the production of sense or antisense nucleic acids.
The subject invention also provides novel methods and compositions for controlling gene expression in plants which utilize the polynucleotide sequences disclosed within the sequence listing. Particularly, the polynucleotides disclosed herein are useful as target genes for the synthesis of antisense RNA or dsRNA useful for RNA-mediated gene interference.
The dsRNA arising from the practice of this aspect of the invention are useful for the study of gene function in vivo, specific down-regulation of a target gene, investigation of gene function, and the discovery and manipulation of biological processes and pathways. By way of example, biosynthetic pathways may be manipulated by the practice of this invention by inactivation of specific gene products, such as enzymes, to allow for the accumulation of intermediate biosynthetic products.
Another use of the ssRNA and/or dsRNA provided by this aspect of the invention concerns the identification of genomic loci useful for the insertion of transgenes in the genome of a plant. In this embodiment of the invention, plants are evaluated using ssRNA and/or dsRNA provided for by this aspect of the invention and examined for phenotypic change. It is well recognized in the art that the incorporation of transgenes into the genome of a transformed plant or animal can result in the inactivation of a gene. When such an event occurs, the transformed plant or animal can suffer deleterious side effects (such as reduced viability, decreased vigorousness of growth, or increased/ uncontrolled cell growth), evidence phenotypic changes, or die. Thus, where the ssRNA and/or dsRNA administered to the plant or animal does not cause death, deleterious effects, or phenotypic changes upon the treated plant or animal, a site for the insertion of a transgene has been identified. Transgenes inserted into these sites would not result in the inactivation of a gene essential to the survival, vitality, viability, or phenotypic characteristics of the transformed animal or plant.